1. INTRODUCTION . . .
2. BACKGROUND OF THE INVENTION . . .
2.1 IMMUNITY AND IMMUNIZATION . . .
2.2 THE IMMUNE RESPONSE . . .
2.3 ADOPTIVE IMMUNOTHERAPY OF CANCER . . .
3. SUMMARY OF THE INVENTION . . .
4. BRIEF DESCRIPTIONS OF DRAWINGS . . .
5. DETAILED DESCRIPTION OF THE INVENTION . . .
5.1 SOURCES OF ANTIGENIC CELLS . . .
5.2 SOURCES OF IMMUNE CELLS . . .
5.2.1 IMMUNE CELLS PRIMED IN VIVO . . .
5.2.2 IMMUNE CELLS PRIMED IN VITRO . . .
5.3 GENERATION OF ANTIGEN-REACTIVE T CELLS . . .
5.4 PREPARATIONS OF HEAT SHOCK PROTEIN-ANTIGEN COMPLEXES . . .
5.4.1 PREPARATION AND PURIFICATION OF HSP 70 PEPTIDE COMPLEXES . . .
5.4.2 PREPARATION AND PURIFICATION OF HSP 90 PEPTIDE COMPLEXES . . .
5.4.3 PREPARATION AND PURIFICATION OF GP96 PEPTIDE COMPLEXES . . .
5.4.4 IN VITRO PRODUCTION OF HSP-ANTIGENIC MOLECULE COMPLEXES . . .
5.5 DETERMINATION OF REACTIVITY OF RESPONDING T CELLS . . .
5.6 REINFUSION OF ANTIGEN-REACTIVE T CELLS . . .
5.7 TARGET INFECTIOUS DISEASES . . .
5.8 TARGET CANCERS . . .
6. EXAMPLES . . .
6.1 MATERIALS . . .
6.2 CHARACTERIZATION OF ANTIGEN-REACTIVE T CELLS
6.3 ANTIGEN RECOGNITION BY CD4+ T CELLS . . .
The present invention relates to methods for generating T cells reactive to an antigenic molecule (antigen-reactive T cells) for use in immunotherapy for the treatment and prevention of cancer and infectious diseases. The methods involve immunizing an animal and incubating the immune cells in vitro with a non-covalent complex of a heat shock protein (HSP) and an antigenic molecule. Methods for pulsing antigen presenting cells and/or immune cells with HSP-antigen complexes for the generation of CD4+ antigen-reactive T cells are provided. Methods and compositions are also provided for the treatment and prevention of cancer or infectious disease in a subject comprising administering to the subject antigen-reactive T cells that are expanded in vitro by the present methods.
2.1 IMMUNITY AND IMMUNIZATION
The immune system protects a host against pathogens by mounting an immune response which is specific to an antigen of an invading pathogen. The objective of immunization is to elicit an early protective immune response by administering to the host an attenuated pathogen, or an antigen associated with a pathogen. This approach has been implemented successfully to prevent a variety of infectious diseases, such as polio, tetanus and diphtheria.
Immunization may be accomplished passively by administering either preformed immunoreactive serum or cells; or actively by presenting a suitable antigenic stimulus to the host""s immune system.
Passive immunization is useful for a host who cannot produce antibodies, or for those who might develop disease before active immunization could stimulate antibody production. However, antibodies produced following some infections, particularly those due to mycobacteria, fungi, and many viruses, are not effective in protecting against the infection. Rather, the action of lymphocytes and macrophages largely determines recovery from these diseases.
Active immunization may be achieved with either viable or non-viable antigenic agents. Viable agents are generally preferred because the immune response provoked is more reliable and long-lived. However, viable vaccines may cause serious illness in an immunologically incompetent host, such as patients receiving corticosteroids, alkylating drugs, radiation or immunosuppressants. The use of attenuated strains always carries the risk that the attenuated agent may recombine with host DNA and mutate into a virulent strain. See generally, Ada, G. L., 1989, Chapter 36, in Fundamental Immunology, 2nd edition, ed. Paul W. E., Raven Press, New York, pp. 985-1032; Cohen, S. N., 1987, Chapter 37, in Basic and Clinical Immunology, 6th edition, ed. Stites, Stobo and Wells, Appleton and Lange, pp. 669-689.
2.2 THE IMMUNE RESPONSE
Cells of the immune system arise from pluripotent stem cells through two main lines of differentiation: a) the lymphoid lineage producing lymphocytes (T cells, B cells, natural killer cells), and b) the myeloid lineage (monocytes, macrophages and neutrophils, as well as accessory cells including dendritic cells, platelets and mast cells). In the circulatory system and secondary lymphoid organs of an adult animal, lymphocytes recirculate and search for invading foreign substances.
Pathogens and antigens tend to be trapped in secondary lymphoid organs, such as the spleen and the lymph nodes, where antigens are taken up or xe2x80x9ccapturedxe2x80x9d by antigen-presenting cells (APCs). The antigen presenting cells serve to display peptides and antigens to the immune cells by placing these peptides on the surface of the APC in association with a major histocompatibility complex (MHC) molecule. The process of antigen capture may occur by phagocytosis of exogenous proteins or by directed transport of proteins within the cell. Alternately, antigens may be derived from proteins synthesized within the cell. Next, antigens are processed into antigenic peptides by proteolytic degradation within the APC. The antigenic peptides are further complexed with a MHC molecule for presentation at the cell surface. Once an antigenic peptide is displayed by an MHC molecule on the antigen presenting cell (APC) surface, a cell-mediated immune reaction may follow which requires an interaction between the APC and a T cell. This interaction can trigger several effector pathways, including activation of T cells, and stimulation of T cell production of cytokines.
Interaction of an APC with a T cell is determined by several major components. These components include a) the T cell surface marker, b) the class of MHC molecule, and c) the T cell receptor (TCR).
T cells can be subdivided by their expression of surface markers CD4 and CD8. T cells expressing CD8 are often known as suppressor or cytotoxic cells. T cells expressing CD4 are often known as helper or inducer T cells. However, the CD8/CD4 dichotomy refers to the pattern of MHC association and antigen recognition. The CD8/CD4 nomenclature does not distinguish between cytotoxic and non-cytotoxic cells. The CD4 molecule binds to conserved structures of the class II MHC molecule. The CD8 molecule binds to conserved structures of class I MHC molecule.
The second factor important in APC/T cell interaction is the MHC. As indicated supra, the CD4 and CD8 molecules bind to the conserved structures of class II and class I MHC molecules, respectively. Class I and class II MHC molecules are the most polymorphic proteins known and play a major role in the immune system in the recognition of self and non-self. The heterogeneity of MHC molecule is observed at the level of haplotype or the combination of classes I and II MHC molecules encoded on a single chromosome. In the human, three distinct genetic loci designated, HLA-A, HLA-B and HLA-C, have been identified encoding class I molecules. Similarly, the three distinct loci encoding class II MHC molecules include HLA-DP, HLA-DR, and HLA-DQ. The multiple loci of MHC genes contribute to the complexity of self and non-self recognition process.
The third component important in APC/T cell interactions is the T-cell receptor (TCR). The TCR is responsible for the antigenic specificity of the T cell, and may only bind antigenic peptide that is associated with the polymorphic determinants of an MHC. Because the binding of the T-cell receptor is specific for a complex comprising an antigenic peptide and the polymorphic portion of the MHC molecule, T cells may not respond or respond poorly when an MHC molecule of a different genetic type is encountered. This specificity of binding results in the phenomenon of MHC-restricted T-cell recognition and T-cell cytotoxicity.
In pathogen-infected cells, proteins of the pathogens are degraded inside the cell. Some of the resulting peptides are transported into the lumen of the endoplasmic reticulum and may form complexes with class I MHC molecules. It has been previously shown that the pathogenic antigens can be chaperoned by heat shock proteins into the endogenous pathway whereby antigenic peptides become associated with the MHC molecules (Suto et al., 1995, Science 269:1585-1588; Srivastava et al., 1994, Immunogenetics 39:93-98). These peptide-MHC complexes are then transported to and accumulate on the cell surfaces, where they are recognized by receptors on T cells (Yewdell et al., 1992, Adv. Immunol. 52:1-123; Bevan, 1995, J. Exp. Med. 182:639-641).
T lymphocytes (T cells) are the critical regulatory and effector cells of the adaptive immune system. T lymphocytes develop and undergo selection in the thymus, and then mature into functional T cells in the tissues after receiving a series of signals. Early signals are triggered by specific antigen-MHC complexes on the surface of antigen-presenting cells (APC). The later signals may be provided by cytokines produced by CD4+ helper T cells, such as interferon-7, and interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7) and interleukin-12 (IL-12).
2.3 ADOPTIVE IMMUNOTHERAPY OF CANCER
Studies with experimental animal tumors as well as spontaneous human tumors have demonstrated that many tumors do express antigens that can induce an immune response. Some antigens are unique to the tumor, and some are found on both tumor and normal cells. Several factors can greatly influence the immunogenicity of the tumor induced, including, for example, the specific type of carcinogen involved, and immunocompetence of the host and latency period (Old et al., 1962, Ann. N.Y. Acad. Sci. 101:80-106; Bartlett, 1972, J Natl Cancer Inst 49:493-504). It has been demonstrated that T cell-mediated immunity is of critical importance for rejection of virally and chemically induced tumors (Klein et al., 1960, Cancer Res. 20:1561-1572; Tevethia et al., 1974, J. Immunol. 13:1417-1423). The cytotoxic T cell response is the most important host response for the control of growth of antigenic tumor cells (Anichimi et al., 1987, Immunol. Today 8:385-389).
Adoptive immunotherapy of cancer takes the therapeutic approach, wherein immune cells with an antitumor reactivity are administered to a tumor-bearing host, with the objective that the immune cells cause either directly or indirectly, the regression of an established tumor. Immunization of hosts bearing established tumors with tumor cells or tumor antigens has generally been ineffective since the tumor is likely to have elicited an immunosuppressive response (Greenberg, P. D., 1987, Chapter 14, in Basic and Clinical Immunology, 6th ed., ed. by Stites, Stobo and Wells, Appleton and Lange, pp. 186-196). Thus, prior to immunotherapy, it has been necessary to reduce the tumor mass and deplete all the T cells in the tumor-bearing host (Greenberg et al., 1983, page 301-335, in xe2x80x9cBasic and Clinical Tumor Immunologyxe2x80x9d, ed. Herbermann RR, Martinus Nijhoff).
Animal models have been developed in which hosts bearing advanced tumors can be treated by the transfer of tumor-specific syngeneic T cells (Mulxc3xa9 et al., 1984, Science 225:1487-1489). Investigators at the National Cancer Institute (NCI) have used autologous reinfusion of peripheral blood lymphocytes or tumor-infiltrating lymphocytes (TIL), T cell cultures from biopsies of subcutaneous lymph nodules, to treat several human cancers (Rosenberg, S. A., U.S. Pat. No. 4,690,914, issued Sep. 1, 1987; Rosenberg et al., 1988, N. Engl. J. Med., 319:1676-1680). For example, TIL expanded in vitro in the presence of IL-2 have been adoptively transferred to cancer patients, resulting in tumor regression in select patients with metastatic melanoma. Melanoma TIL grown in IL-2 have been identified as activated T lymphocytes CD3+ HLA-DR+, which are predominantly CD8+ cells with unique in vitro antitumor properties. Many long-term melanoma TIL cultures lyse autologous tumors in a specific class I MHC molecule and T cell antigen receptor-dependent manner (Topalian et al., 1989, J. Immunol. 142:3714).
Application of these methods for treatment of human cancers would entail isolating a specific set of tumor-reactive lymphocytes present in a patient, expanding these cells to large numbers in vitro, and then putting these cells back into the host by multiple infusions. However, the methods of Rosenberg for generating tumor-reactive lymphocytes require the use of intact irradiated tumor cells with potential broad antigen specificity, as a source of stimulation of lymphocytes. Additionally, since T cells expanded in the presence of IL-2 are dependent upon IL-2 for survival, infusion of IL-2 after cell transfer prolongs the survival and augments the therapeutic efficacy of cultured T cells (Rosenberg et al., 1987, N. Engl. J. Med. 316:889-897). However, the toxicity of the high-dose IL-2 and activated lymphocyte treatment has been considerable, including high fevers, hypotension, damage to the endothelial walls due to capillary leak syndrome, and various adverse cardiac events such as arrhythmias and myocardial infarction (Rosenberg et al., 1988, N. Engl. J. Med. 319:1676-1680). Furthermore, the demanding technical expertise required to generate TILs, the quantity of material needed, and the severe adverse side effects limit the use of these techniques to specialized treatment centers.
Despite the teachings of Rosenberg, severe deficiencies exist in the art regarding methods of cellular immunotherapy. In many instances, it is not possible to generate tumor cell line. Thus, it would be desirable to have a method for generating a large number of activated/stimulated T cells reactive to any antigen or a large repertoire of antigens without reliance on intact tumor cells, which would have the convenience of in vitro culture.
The process of T cell priming is poorly understood for most purposes. There have been occasional reports of priming of antigen-specific T cells in vitro (Steel and Nutman, 1998, J. Immunol. 160: 351-360; Tao et al., 1997, J. Immunol. 158:4237-44; Dozmorov and Miller, 1997, Cell Immunol. 178:187-96; De Bruijn et al., 1991, Eur J Immunol. 21:2963-2970; De Bruijn et al., 1992, Eur J Immunol. 22:3013-3020; Houbiers et al., 1993, Eur J Immunol. 26:2072-2077; Nair et al., 1997, Eur J Immunol. 27:589-597), however, they are restricted to instances where a higher expression of a given type of MHC I-peptide complex De Bruijn et al., 1991, Eur J Immunol. 21:2963-2970; Nair et al., 1997, Eur J Immunol. 27:589-597) or a particularly high avidity for the MHC I-peptide-T cell receptor interaction has been achieved (De Bruijn et al., 1991, Eur J Immunol. 21:2963-2970).
The present invention relates to methods for generating antigen-reactive T cells in vitro that can be used for the prevention or treatment of a disease or disorder, such as infectious disease or cancer. In one embodiment, the present invention relates to methods for generating antigen-reactive CD4+ T cells that can be used for the prevention or treatment of a disease or disorder, such as infectious disease and cancer. The methods of the invention provide CD4+ T cells that are capable of specifically killing or targeting antigenic cells, such as cancer cells or infected cells comprising an antigen with which the T cell has been stimulated.
The invention provides methods for generating T cells reactive to an antigen comprising immunizing an animal and incubating the immune cells in vitro with a non-covalent complex of heat shock protein and an antigenic molecule. In various embodiments, the invention provides immune cells used in the methods of the invention that are enriched for CD4+ T cells or antigen presenting cells.
Alternatively, in another embodiment immune cells that are primed/immunized in vitro can also be used. In various embodiments, antigenic cells are used as a source of antigenic molecule and/or HSP-antigen complex by the methods of the invention. The antigenic cells can be cancer cells or cells infected with a bacteria, fungus, parasite, or a protozoan. The invention further provides for the use of antigenic cells which expresses recombinant antigenic molecule or which have been infected in vitro with a pathogen or which have been transformed in vitro.
The invention provides the use of HSP-antigen complexes comprising a heat shock protein which is non-covalently complexed to an antigenic molecule. Specifically, the invention provides for the use of HSP60, HSP70, HSP90, HSP100, gp96 or a member of the small heat shock protein (HSP) family.
Methods are provided for stimulating and/or restimulating immune cells with a non-covalent complex of an HSP and an antigenic molecule. In one embodiment, the immune cell stimulation and/or restimulation result in antigen-reactive CD4+ T cells.
The invention provides methods of recovering antigen-reactive T cells from culture.
Further, the invention provides methods of treating or preventing a disease or disorder in a subject comprising generating T cells reactive to an antigenic molecule by a method of the invention and administering an effective amount of the antigen-reactive T cells to the subject. In a preferred embodiment, the T cells and antigenic cells have at least one MHC allele in common. In a more preferred embodiment, the T cells and the antigenic cells have more than one MHC allele in common. In most preferred embodiment, the method of the invention uses immune cells and antigenic cells from the same human.